System for environmental microbial testing

ABSTRACT

A gelled biological growth medium in a culture container, as well as kits and methods of producing the growth medium, utilize gelling of a liquid growth medium containing low methoxyl pectin on a film of calcium salt deposited on a growth surface of the culture container. The salt acts as a trigger for gelation and is conveniently deposited on the walls by evaporation of methanol or another volatile solvent.

BACKGROUND

Methods for controlled cultivation of microorganisms on the surface ofgelled media are in widespread use. Techniques have been developed tostudy bacteria, fungi, mosses, and eukaryotic cells and tissues. Gelledmedia are also used in environmental microbial testing.

Awareness of environmental hazards has led to an increased desire toconduct microbial testing, and microbial test kits are available forsuch testing. For practical microbial field testing, it has becomenecessary to conduct such testing without the use of heating and/orlaboratory sterilization of culture media in the field, and without theuse of equipment such as autoclaves, glove boxes, or biological hoods inthe field.

Agar is a commonly utilized gelling material for microbial culturing inPetri dishes and the like. Agar is derived from algae and includes amixture of the polysaccharide agarose together with agaropectin. Agarhas been used for many decades to solidify nutrient-containing growthmedia used for culturing and identifying different species of bacteria,yeasts and molds. A suspension of agar powder in liquid nutrient mediumis typically boiled to dissolve the constituent polysaccharides,sterilized, and then cooled to approximately 50-60° C. before pouringthe liquid medium into a culture container such as a glass orpolystyrene Petri dish. The preparation of a sterile agar Petri dishtypically requires specialized skill in sterile techniques and inpreparation of the agar or other suitable growth media.

The availability of simplified and more rapid preparation methods forgelled culture media would improve the convenience and utility ofmicrobial testing in the field such as in residential homes,manufacturing facilities and businesses, especially when performed bypersons without specialized skills or equipment.

SUMMARY

The present technology relates to the field of sterile biologicalculture media and provides methods and kits for making an easily andrapidly gelled sterile culture medium, that includes pectin as thegelling agent. Final preparation and sterile provision of a microbialsampling and culturing container with culture medium can be performedwithout access to special equipment or techniques, and can be performedwithout specialized training or equipment.

A pectin-based gelling system providing a source of calcium ions to geland solidify a nutrient medium obviates the need to boil or otherwiseheat or reheat a cell culturing medium immediately before pouring themedium into a Petri dish. Therefore, it is well suited for field workand consumer use where controlled heating/sterilization may beimpossible or inconvenient. The present technology provides a system forproduction of such growth media, the system for microbial analysis andoversight by those with skill in the art, where the growth media can bedistributed to end users who possess ordinary skill, the growth mediaprovided in a kit with instructions. The present technology moves thepreparation of biological growth media to the hands of more users byproviding sterile methods for producing gelled culture media that can befollowed quickly and, for example, in remote locations.

The present technology provides a kit. The kit overcomes difficulties inpreparing biological growth media, for example, sterilization of thecontainer, sterilization of the medium, transportation and assembly ofthe biological growth system, time involved, and necessary end-userskill. Final preparation of the biological growth medium from the kitinvolves no specialized skill in microbiology or in the preparation ofsterile growth media. Instead, persons preparing the biological growthmedia follow general methods and instructions described therein. Thepresent technology also involves methods of preparation of biologicalgrowth media and methods of using the prepared media includingspecialized containers designed for use.

The present technology utilizes pectin for gelling of growth media, andno agar is needed. An aspect of the system is the preparation andsterilization of a growth media containing amidated low methoxy pectinalong with nutrients. While unmodified low methoxy (LM) pectin can beused in the present technology, the use of a chemically modified LM lowester pectin that has been altered by amidating the pectin isbeneficial. Such an amidated pectin is sometimes referred to as an LApectin or even an LMA pectin. The amidated pectin molecules are modifiedby converting some of the galacturonic acid residues to carboxylic acidamides (e.g., using ammonia) and the resulting pectins can form stronggels over a wider range of calcium concentrations than unmodified LMpectins.

In one example, these LA pectin soluble solids are contributed indissolved potato dextrose broth or “PDB” (obtained from AlphaBiosciences, Baltimore, Md.) that includes 2% dextrose and 0.4% potatoextract, where as little as 2% by weight of added LA pectin issufficient to solidify the broth using approximately 15-60 mg calciumper gram of pectin without substantial syneresis (release of free liquidfrom the gel) occurring. Two examples of amidated low ester pectins thathave been successfully used at a level of 2% by weight pectin to formgelled PDB nutrient growth medium herein include LA 410 pectin obtainedfrom DuPont Danisco USA (New Century, Kans.) and amidated low methoxypectin LM 35 obtained from TIC Gums, Inc. (White Marsh, Md.). Whilethese examples do not limit the present technology, many more suppliersof amidated low ester pectins are available.

In another example, chloramphenicol (IBI Scientific, Dubuque, Iowa) isalso included as a broad spectrum antibiotic agent at a level ofapproximately 50-100 micrograms/milliliter in the pectin-containing PDBnutrient growth medium for inhibiting bacterial growth and selectivelydetecting the presence of airborne mold spores.

In another example, the pectin-containing growth medium can be producedwith specific nutrients to promote growth of targeted cells or, as inthe example above, specific agents to prevent growth of undesiredbiologics. The kits produced by the system described herein can betargeted to selectively cultivate microbes or other cells or tissues,e.g., bacteria or molds or cells or tissues of higher eukaryotes, andthe growth medium contained therein can be tailored for the needs of theend user.

Typically, LM pectins may be gelled using between approximately 15 mgand 30 mg of calcium per gram of pectin. However, between 10 and 60 mg,or even as high as 90 mg, calcium per gram of pectin can be used in theLA gels, containing surprisingly low levels of dissolved solids, withoutexcessive syneresis occurring even 1-2 days after forming the gel.Syneresis would normally be expected to occur as calcium ions causecontraction of the gel as pectin polysaccharide molecules are drawncloser together by calcium ions. Syneresis in a Petri dish isproblematic in some examples. When attempting to detect and count thenumber of environmental mold spores or bacteria settling on a solidculturing medium, free water on a solid culturing medium can allowmultiplying mold cells and also bacteria cells to spread away from theirinitial contact point and then confuse the measurement. Syneresis wouldbe expected to be particularly problematic when only low levels ofdissolved solids, a.k.a., soluble solids, are present in the gel. Forexample, in some nutrient gels produced herein for culturing anddetecting mold spores the gels contain as little as 2.4% by weight ofsoluble solids.

Using the production methods disclosed herein, the growth medium withpectin is placed in a secure package after preparation. Sterilization ofthe growth medium can be done before, during, or after packaging, usingsterilization techniques known in the art. The package is suitable forstorage and transportation, and the sterilized growth medium does notgel until it comes into contact with a gelling agent, which containscalcium. While the package of growth medium is suitable for storage andtransportation, the package is preferably supplied within a kit, and thekit can be packaged to further ensure stability duringstorage/transportation. Alternatively, the entire kit can be sterilizedafter assembly but before shipping, for example, by irradiation,pasteurization, or microwaves.

Using the methods of production disclosed herein, a solution of calciumlactate is prepared in methanol, and the methanol sufficientlysterilizes the solution. Enough methanol is used to dissolve the calciumlactate. The calcium provided by this solution is utilized as a gellingagent for the pectin-containing growth media. This gelling agentsolution is securely packaged and is suitable for storage andtransportation. Alternatively, other alcohols such as ethanol orisopropanol may be added to the methanol solution of calcium lactate butin proportions to preferably not cause precipitation of the calciumlactate. The package of gelling agent is preferably supplied within akit that includes a cell culture container, along with the package ofgrowth media described above, and the kit can be packaged to furtherensure stability during storage/transportation. If other soluble organicsalts of calcium are utilized to form a film in the cell culturecontainer, the salts may dissolve and be either moderately or highlysoluble in methanol, or alternatively the organic salts may form aslurry with the organic salts being either poorly, moderately or highlysoluble in methanol or other volatile solvent, and the slurry stillperforms the intended function(s).

In an embodiment, the kit contains a package of pre-sterilized pectinmedium, a package of pre-sterilized calcium lactate in methanolsolution, a Petri dish or another container with lid all pre-sterilized,and instructions for use. In some cases, instructions/methods areincluded with the kit or printed on the container(s). In other cases,instructions are transmitted by email, website, text message, or otherelectronic means. The end user of the kit follows the instructions andcan quickly cause the gelling of a sterile pectin-containing growthmedium using the kit and method provided.

Another aspect of the present technology is a method of use wherein theperson conducting the microbial sampling, e.g., exposure to air or waterby the end user, follows instructions so that a container ofpre-sterilized calcium lactate solution in methanol is opened and pouredinto a pre-sterilized Petri dish or similar container. The end userensures that at least the bottom interior surface of the Petri dish orcontainer is evenly covered by the solution. The end user then waits forthe methanol to evaporate, at least depositing a consistent film ofcalcium lactate upon the bottom of the container. Then thepre-sterilized pectin-containing growth medium is poured into the Petridish or similar container, and the lid to the container is applied.After a brief time interval, the pectin forms a gelled growth mediumthat is ready for use, e.g., ready for exposure to air, water samplesand/or other materials containing possible microbial contaminants.Formation of the gel can be established by picking up the container inthe hand and noting that the gel does not tilt within the container.This method of the present technology enables an end user to prepare asterile solidified growth medium far from sterilization equipment suchas autoclaves, glove boxes, or biological cabinets.

In an embodiment, the system of the present technology produces a kitcontaining a package of pre-sterilized pectin medium, a packagecontaining a Petri dish or similar container, with the Petri dishalready containing a dry uniform internal coating of calcium lactate, atleast upon a portion of the container, all pre-sterilized, andinstructions for use. In this embodiment, the end user of the kit opensthe Petri dish, pours the pectin-containing growth medium into the dish,places the lid upon the dish, and waits a brief time, e.g., 15-30minutes for a gel to form. The uniformity of a dried coating (preferablyin the form of a precipitate or film deposited on a growth surface ofthe dish) of calcium lactate in a Petri dish or other culture container,that assures the uniformity of pectin gel formation over the cell growthsurface of the dish, can be visually monitored before adding the growthmedium. This monitoring can be facilitated by a white translucent orsemi-opaque appearance of the dried adherent calcium lactate coating onthe dish. If any area on the dish has been poorly or deficiently coated,that area appears more transparent/less opaque. Where methanol has beenused to solubilize the calcium lactate, the white appearance of acalcium lactate coating can be adjusted by including a small amount ofwater, e.g., approximately 2%-4% by volume, in the methanol as thecalcium lactate crystallizes on the surface of a cell culture container.It is believed that this level of moisture provides crystalline water ofhydration that enables some or most of the calcium lactate toprecipitate in the form of calcium lactate pentahydrate crystals thatscatter light and thus appear white. By comparison, an anhydrous calciumlactate coating that is precipitated from anhydrous methanol produces anearly invisible clear transparent coating on the surface of a Petridish or other cell culture container.

In embodiments, the manufactured kit can be sold at retail to those withordinary skill who seek to perform microbial testing or who, forexample, seek to cultivate bacteria, moss, or fungi on a medium. Inanother example, the kits are produced by a quality assurance group anddistributed to hospitals in various locations for use by qualitysampling groups. The kits produced herein are durable for transportationand storage. Potentially the kits described herein could be shipped tofar reaches for specific explorations.

In an embodiment, the disclosed system involves calculation of an amountof pre-sterilized pectin media, sufficient for a certain size container.The system also involves preparation of pre-sterilized calcium lactatesolution in methanol, in amount sufficient to gel the pectin media andto coat, at least the bottom interior surface of, a certain sizecontainer, and placement of this solution into a sealed container forlater use. The system also involves secured placement of apre-sterilized or cleaned Petri dish or similar container, with knowninternal area, with lid onto container, for example a sealed box ofsuitable material, along with the container of pectin media andcontainer of calcium lactate solution, sealing the box, and transportingor shipping the box to the person conducting the microbial testing.

In an alternative embodiment, only the package of pre-sterilized pectingrowth media and the package of pre-sterilized calcium lactate solutionis shipped to the end user conducting the microbial testing, and thisperson already has suitable sterile containers with lids. Instructionsfor use could be provided with the packages, printed on labels, or sentby e-mail or other electronic means.

The persons using the kit disclosed herein can conduct microbial testingor biological cultivation and can follow general instructions forpreparation of the microbial testing kit and subsequent brief orcontrolled exposure of the gelled medium to the environment such asexposure intervals of approximately 10 min to 4 hr, or approximately 30min to 2 hr, or approximately 1 hr. For example, the kit may travel witha health inspector, who uses the kit on-site to sample food from avendor or water from a drinking well. Potentially an archeologistobtains the kit to sample microbes from a sealed location that has notbeen unsealed for thousands of years. The present technology uniquelyprovides a kit that can be opened and can provide sterile microbialtesting media in a remote environment in a short time, surprisingly,even after the kit has been in transportation and/or storage for aconsiderably long time.

If desired, the shelf-life of the kit discussed herein can be extended,for example, by secured sterile packaging, use of ultra-high-temperatureprocessing during manufacture, irradiation, additives, or other meansknown in the art. The system disclosed herein also includesdetermination of kit shelf-life, if desired, by stability testing undervarious accelerated conditions and/or subsequent prediction ofshelf-life using techniques known in the art.

The liquid growth medium may contain liquid growth medium with otheradded ingredients, for example, preservatives, blood cells, pHindicators, neomycin, beef broth, gentamicin, indicators, nutrients,soy, and buffers.

One experienced in the art will recognize the significance of thepresent technology in expanding the provision of growth media to endusers, and it is understood that embodiments of the present technologyencompass various systems, methods, and physical constructions, withinthe idea of the technology. The system, method(s) and embodiment(s) arefurther described below.

The covered or uncovered growth container, in an embodiment, contains aninternal surface coating or film of calcium lactate deposited fromevaporation of methanol, the surface coating or film at leastsufficiently coating the bottom interior surface of the container, thecontainer suitable for gelling of a liquid growth medium containing lowmethoxyl pectin or amidated low methoxyl pectin material, with, forexample, the coating or film of calcium lactate able to be convertedfrom anhydrate forms to hydrate crystalline forms or and/or proportionsof various hydrated forms without affecting the suitability for gellingof the liquid growth medium.

The present technology, in an embodiment, is a calcium lactate film(consisting essentially of a solid form of calcium lactate saltdeposited as a layer) adhered to a transparent plastic or glass surface,wherein said film is cast on said surface by evaporation from a solutioncomprising calcium lactate and at least some methanol. This film isremarkable in being durable and tightly bound to the surface (eitherglass or polystyrene or polylactide thermoplastic, for example) in whichthe film can be transparent or translucent yet white or whitish incolor. The film may or may not contain crystals of calcium lactatevisible with a microscope or the unaided eye. If some water is presentin the methanolic calcium lactate solution, the resulting film iswhitish translucent. If the calcium lactate solution is anhydrousmethanol, the resulting film is transparent. The film is useful inslowly dissolving and releasing free calcium ions (i.e., divalentcations of calcium) into a nutrient cell culture medium comprising adissolved calcium-reactive pectin that can be gelled by the calciumions. In this regard, the film containing a calcium salt, such ascalcium lactate, is preferably not present in or embedded in anothermaterial, such as a gel, polymer matrix, a plurality of microparticlesor nanoparticles, or a water insoluble material or coating, but ispresent as a salt material substantially free, essentially free, orentirely free of other materials, particularly water-insolublematerials, so as to promote optimal dissociation of the salt and releaseof calcium ions when a gellable growth medium is added to the culturecontainer and contacts the calcium salt film. This embodiment of thetechnology can be provided to the end-user along with sterilepectin-containing growth media and instructions, all in a kit.

The method of preparation, in an embodiment, is performed wherein saidliquid growth container is a Petri dish and the amount of a volatilesolvent liquid is between 0.5 mL and 2 mL per 90 mm diameter(approximately 64 cm² bottom interior surface area) Petri dish,equivalent to between 0.0078 mL and 0.0313 mL per square centimeter ofPetri dish area, or between 0.005 mL and 0.050 mL per square centimeterof Petri dish area, or between 0.001 mL and 0.100 mL per squarecentimeter of Petri dish area, or wherein the entire interior surfacearea of the Petri dish is calculated in place of the surface area of thebottom interior surface of the Petri dish and substituted for theabovementioned value ranges.

Ensuring that about 2% to 5% water is contained in the methanol solutionof calcium lactate has been found to be beneficial because the presenceof this small amount of water results in the calcium lactate coatingsdrying with a whitish translucent appearance. The whitish translucentappearance is beneficial in confirming that the calcium lactate coatingis uniform across the entire surface of the Petri dish or othercontainer. Dried coatings of calcium lactate from anhydrous methanolicsolutions are transparent and more difficult to monitor for coatinguniformity.

The volume percentage of methanol can vary, for example, within therange of volume percentages selected from the group consisting ofbetween 1% and 100% methanol, between 7% and 100% methanol, between 50%and 100% methanol, between 90% and 100% methanol, and between 99% and100% methanol.

The amount of calcium lactate is variable, for example, and is withinthe range of between approximately 25 mg and 100 mg anhydrous calciumlactate per 0.3 g of low methoxyl pectin in approximately 15 mL of saidliquid growth medium, equivalent to between approximately 85 mg and 350mg anhydrous calcium lactate per gram of said pectin.

The amount of calcium lactate is within the range of betweenapproximately 25 mg and 100 mg, or as high as 200 mg, of anhydrouscalcium lactate per 0.3 g of low methoxyl pectin in approximately 15 mLof said liquid growth medium, equivalent to between approximately 85 mgand 350 mg, or may be as high as 700 mg, of anhydrous calcium lactateper gram of said pectin.

The kit, method, or system described herein, can also be embodiedwherein the end user sends the biologic, which has grown upon theculture media, back to the originator of the kit for analysis.

The kit may enable preparation of the pectin-gelled sterile growthmedium from the kit in which preparation may be in any of a variety ofharsh environments. Accordingly an alternative embodiment is the kit,method, or system described herein, wherein a pre-formed calcium lactatefilm is provided, for example, within a flexible growth container, andthe packaged growth medium is released within the growth container byapplying pressure to the flexible growth container. In this example thepackaging is ruptured by external pressure on the growth container bythe end user. In another embodiment, the package or packages are openedexternal to the container by pressure or by breaking only a portion ofthe package. The growth medium and the calcium lactate solution may bothbe contained in rupture packages. The presently disclosed technologyencompasses various forms of packaging and is not limited to thepresently disclosed packages.

The user may wait 5 to 60 minutes, 10 to 40 minutes, 10 to 20 minutes,15 to 30 minutes, 20 to 45 minutes, 20 to 25 minutes, 15 to 20 minutes,30 to 120 minutes, or 40 to 180 minutes for the growth medium to gel. Ina normal ambient environment, pectin-containing cell culture mediadescribed herein can gel to a non-fluid useful state for physicalhandling when added to a calcium lactate-coated Petri dish in as littleas 10-20 or 10-30 minutes. Alternatively, the user may wait for a periodof time that is not pre-determined due to, for example, unforeseenenvironmental conditions or temperatures in a harsh environment.

In a method described herein, the calcium lactate can be replaced byanother salt, for example, a fine powder of calcium fumarate anhydrous,calcium malate anhydrous, or calcium gluconate. Preferably, thesubstitute salt forms a slurry/suspension with a volatile organicsolvent, such as methanol. If a slurry is used, the Petri dish orcontainer containing the slurry can be tilted back and forth by the enduser to uniformly coat the bottom of the Petri dish or container beforeallowing the slurry to dry. If a slurry is used, the culture medium maytake longer to gel and can take, for example, from 1 to 2 hours.Therefore, calcium lactate may be partially/wholly replaced by anotherorganic salt of calcium or a combination of calcium salts, so long asthe purpose of providing a container of sterile gelled growth medium isachieved.

The methods and kits described in any of the above embodiments cancomprise any organic calcium salt that is sufficiently soluble, i.e.,highly or moderately soluble in a volatile solvent, for example,methanol or other volatile solvent, with or without calcium lactate, ormay form a slurry in a volatile solvent.

The kit, method, or system described herein may include or embody any ofthe following methods or processes wherein the end-user applies thecalcium lactate solution to the container on a flat surface and waitsfor methanol evaporation, wherein the end user applies the calciumlactate solution to the container and gently moves the container toensure an even coating of calcium lactate solution, wherein the end userapplies the calcium lactate solution to the container and applies heator air to aid solvent evaporation or other purposes, or wherein the enduser applies the calcium lactate solution by use of the packagingprovided. In alternative embodiments, a different organic salt ofcalcium is utilized, and the end user applies a slurry of the organicsalt to the container and evenly distributes the slurry.

Calcium lactate salt can be used in anhydrous form or as a hydrate.Additionally, the presently disclosed technology encompasses allpossible isomers of calcium lactate or other organic calcium salts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of an adherent calcium lactate film, formed byevaporation of a methanol solution containing 100 mg solubilizedanhydrous calcium lactate and 4% by weight water on the interior of a 90mm diameter Petri dish.

FIG. 2 is a photo of an adherent coating of 100 mg anhydrous calciummalate powder, deposited by evaporation of a methanol slurry of the saltparticles on the interior of a 90 mm diameter Petri dish.

FIG. 3 is a photo of an adherent coating of 200 mg anhydrous calciumgluconate powder, deposited by evaporation of a methanol slurry of thesalt particles on the interior of a 90 mm diameter Petri dish.

DETAILED DESCRIPTION

The present technology provides a convenient and effective method forpreparing gelled growth media for culturing microorganisms. Thetechnology makes use of the ability of pectins to gel in the presence ofcalcium ions, and also benefits from the discovered ability of at leastone water-soluble yet non-hygroscopic calcium salt, calcium lactate, toform a fine, adherent coating on a culture vessel surface whenprecipitated by evaporation from a solution containing the calcium saltdissolved in a volatile organic solvent, e.g., methanol.

Pectins are rich in galacturonic acid residues, many of which arenaturally ethyl-esterified. During purification from vegetable materialssuch as citrus peels and pulp, some of the methyl ester groups are lost,resulting in a mixture of so-called high methoxyl (HM) and low methoxyl(LM) pectins. With LM pectins the degree of methoxylation(esterification of galacturonic acid carboxyl groups) is less than 50%.Typically, about 5%-49% or more, such as about 25%-40%, of thegalacturonic acid residues remain esterified, and the methoxyl contentby weight of the LM pectin is between approximately 2% and 8% or lessthan 7% and often about 5% by weight.

A pectin-based gelling system utilizing calcium ions that can gel andsolidify a nutrient medium at ambient temperature obviates the need toboil or otherwise heat or reheat a microbial culturing medium, such asone containing agar, immediately before pouring the medium into a Petridish or similar container. The system of the present technology providesfor sterile packaging of the pectin-containing medium after production.Therefore, the present technology is well suited for field work,consumer use, and lab use where controlled heating may be impossible,inconvenient, or simply take too long. The sterile package of medium canbe opened at the testing site. It is known that LM pectins may be gelledusing between approximately 15 mg and 30 mg of calcium ions per gram ofpectin. For example, calcium chloride (containing approximately 36% byweight calcium), when used to promote pectin gelation in other systems,requires from about 42 mg to about 84 mg of CaCl₂) per gram pectin forgelling an aqueous medium.

While an unmodified LM pectin can be used, the use of a chemicallymodified LM low ester pectin that has been altered by amidating thepectin (an LA or LMA pectin) can be beneficial. The amidated pectinmolecules are modified by converting some of the galacturonic acidresidues to carboxylic acid amides (e.g., using ammonia) and theresulting pectins can form strong gels over a wider range of calciumconcentrations than the unmodified LM pectins. The percentage ofgalacturonic acid residues converted to carboxylic acid amides, i.e.,the “degree of amidation” for pectins useful herein generally rangesfrom approximately 10% to 25%. For example, LA 410 has a degree ofamidation of approximately 19% and a degree of esterification of theacid groups of approximately 31% while LM 35 has a degree of amidationof approximately 15% and a degree of esterification of approximately35%. It was found that a surprisingly wide range of calcium levels ofbetween 10 mg and 60 mg or even as high as 90 mg calcium per gram ofpectin can be used to gel the LA low ester amidated pectin gels, withsurprisingly low levels of dissolved solids, without excessive syneresisoccurring even 1-2 days after forming the gel.

Use of unmodified LM pectin can create issues with syneresis when onlylow levels of dissolved solids, a.k.a., soluble solids, are present innutrient gels and the gels are shipped, in gelled form, causingsyneresis. While unmodified LM pectin can be utilized in the presenttechnology, advancement in the field of modified pectin is encompassedin the presently described technology. Therefore, the embodimentsdescribed herein do not limit the scope of the presently describedtechnology to the modified pectins currently available, but the presenttechnology anticipates that future pectins could cause firm, durablegelling with less or more calcium content. The chemically modified LA orLMA pectins are preferred.

The growth media described herein containing low methoxyl or amidatedpectin is not limited in the other ingredients and may contain otheringredients such as, for example, preservatives, blood cells, pHindicators, inhibitors, neomycin, beef broth, gentamicin, indicators,soy, and buffers. The growth media described herein can be targeted forgrowing a specific type of organisms and might include nutrients forthat organism while including inhibitors for other types of organisms.

In addition to discovering the advantage of using calcium lactate on asurface with an amidated low ester pectin to limit or fully prevent gelsyneresis in Petri dishes or containers containing a pectin-gelledgrowth medium with a low level of dissolved solids, the presenttechnology provides a convenient and more cost-effective method forintroducing sterile calcium ions into a pectin-containing culture mediumcontained in a Petri dish. Another surprising result of the presenttechnology was to find a method to produce a firm and smooth pectin gelin a short time period.

During research, for example, calcium chloride and calcium nitrate weredissolved in a number of different solvents that were applied directlyto the interior surface of a Petri dish. To achieve more rapid dryingand depositing of the above calcium salts in the bottom of a Petri dish,the salts were dissolved in 1 mL of denatured alcohol. Small quantities(e.g. 50 mg) of these salts were dissolved and then applied directly tothe bottom interior surface of 15 mm×90 mm polystyrene Petri dishes andair dried. As the alcohol solvents evaporated, these calcium saltcoatings dried as irregular spots and streaks on the bottom of thedishes, whereupon the deposited salts soon appeared visibly wet from theuptake of ambient moisture (these salts being hygroscopic ordeliquescent), making the entire procedure problematic. Thereafter, when15 mL of sterile potato dextrose broth (PDB) nutrient medium containing2% by weight dissolved LA 410 pectin (together abbreviated “PDB-P”) waspoured into these 90 mm diameter Petri dishes (coated with theabove-described calcium salts) the PDB-P medium immediately gelled inirregular clumps. It is believed that the deposited spots ofconcentrated (inorganic) calcium salts caused solubilized pectin to gelin a rapid and unpredictable manner. It was concluded that unless acalcium salt could be dried and firmly attached to a Petri dish surfaceand then released and dissolved slowly into the pectin-containingmedium, a smooth gel could not be obtained.

After many attempts at controlling the gelling of a pectin-containingculture medium, it was surprisingly found that an edible calcium saltknown as calcium lactate (Jost Chemical Company, St. Louis, Mo.) quiteremarkably did not dissolve in ethanol but instead dissolved inmethanol. It was also surprisingly found that a limited number of otherorganic salts of calcium could work with the present technology.

Surprisingly, when approximately 1.0 mL to 1.3 mL (or a range of volumefrom approximately 0.7 mL to 1.5 mL) of methanol solutions containingeither 50 mg, 75 mg, 100 mg, or 200 mg of the dissolved anhydrouscalcium lactate was applied so as to coat the bottom of 15 mm×90 mmpolystyrene Petri dishes (Corning, Inc.) by tilting the dishes back andforth, the calcium lactate solutions dried as smooth and adherentuniform films. These films appeared nearly colorless and transparent inthe absence of moisture or alternatively whitish and translucent(visually microcrystalline) when small amounts of moisture were presentin the methanol. Advantageously, owing to the natural disinfectantproperty of primary alcohols including methanol (as well as ethanol andisopropanol for example), the solution of calcium lactate in methanolwas self-disinfecting as were the Petri dish surfaces coated with thesesolutions. This disinfecting property was advantageous in obviatingadditional sterilization steps for the calcium lactate solution and/orthe calcium lactate-coated Petri dish surfaces. Smaller and larger Petridishes, e.g., 30 mm to 150 mm diameters, and microbe culture containersof other shapes and sizes can be conveniently coated withmethanol-containing calcium lactate solutions as described above byarithmetically scaling the quantity of calcium lactate solution to beused according to the surface area to be coated.

Surprisingly, the calcium lactate, after adhering to the Petri dishafter evaporation of the methanol, was not hygroscopic or deliquescentbut instead was stable and durable. Therefore, an embodiment of thepresent technology is a Petri dish or other container with an internalcoating of calcium lactate, at least upon the bottom interior surface,with the calcium lactate coating produced through evaporation ofmethanol or other volatile solvents such as blends of ethanol andmethanol.

In a preferred embodiment, ensuring the presence of or adding about 2%to 5% by volume water to methanolic solutions of calcium lactate isdesired. The presence of this small amount of water in the methanolicsolution results in the calcium lactate coatings drying with a whitishtranslucent appearance. This is beneficial in confirming that thecalcium lactate coatings are uniform across the entire surface of thePetri dish (or other cell culture container) and assures that pectin gelformation will also be uniform. By comparison, dried coatings of calciumlactate from anhydrous methanolic solutions are transparent and moredifficult to monitor for coating uniformity.

The stability of the adherent calcium lactate film was surprisinglyuseful. When 15 mL of the same PDB-P liquid culture medium describedabove was either immediately or even weeks later applied over thesedried films containing the above-cited 50 mg, 100 mg and 200 mgquantities of calcium lactate contained in 90 mm diameter Petri dishes(approximately 64 cm² bottom interior surface area), the films remainedattached to the polystyrene (or alternatively glass) Petri dishes andslowly dissolved into the overlaid liquid medium (15 mL/64 cm² orapproximately 0.25 mL per square centimeter) after several minutes timehad elapsed. Within approximately 15 minutes the pectin-containingculture medium appeared to be gelled and within approximately 30 minutesthe medium had firmly gelled as evident upon finger contact.

Accordingly, an embodiment of the present technology is a Petri dish orsimilar container containing a layer/film of calcium lactate, depositedby the evaporation of methanolic solution, at least upon the bottominterior surface, with the precoated Petri dish or container sealed andsupplied in a kit with a package of pectin-containing growth media. Inthis embodiment, the pectin-containing growth media is pre-sterilized,the package is opened, and the growth media is poured into the Petridish or similar container containing the layer of calcium lactate. Thelid to the container is optionally applied. The calcium lactate slowlydissolves into the growth medium, causing the growth medium to gel. Thepresent technology also encompasses a method of manufacturing a Petridish or other container with said layer/film. The kits containing Petridishes or containers with preformed calcium lactate precipitate coatingsthereby obviate the need for the consumer or end user to handle and pourthe methanolic solution and to form the dried coatings. Methanol istoxic, albeit this is likely a miniscule amount of methanol, and thepreformed coating avoids risks of spilling the methanol solution orforming a poor calcium lactate coating that would result in poor qualitypectin gel.

All four levels of calcium lactate, discussed above, enabled fullgelling of 2% by weight solutions of pectins LA 410 and LM 35 dissolvedin PDB culture media. After 24 hours incubation at room temperature (23°C.) the four different levels of anhydrous calcium lactate (50, 75, 100,and 200 mg) provided in 15 mm×90 mm diameter Petri dishes resulted indifferent amounts of syneresis as quantitated by blotting and weighingfree liquid present around the circumference of the 15 mL gelled culturemedia in Petri dishes. For example, 15 mL of PDB-P medium gelled with 50mg calcium lactate released essentially no free liquid (zero syneresis)after incubation at room temperature for 24 and 48 hours. By comparison,75 mg calcium lactate-gelled PDDB medium released approximately 0.15 to0.25 mL liquid while 100 mg calcium lactate-gelled PDB medium releasedapproximately 0.5 to 1.0 mL liquid and 200 mg calcium lactate-gelled PDBmedium released approximately 2.2 mL liquid based upon duplicate Petridishes.

Therefore, 50 mg and 75 mg of anhydrous calcium lactate-gelled growthmedium (15 mL) released little if any liquid by syneresis and areconvenient usage levels for a Petri dish having an approximate diameterof 90 mm (64 cm² area or about 10 square inches). With the knowledgethat anhydrous calcium lactate contains 18.4% by weight calcium, andwith each Petri dish containing 0.3 g pectin (2%×15 mL) the Petri dishescontained approximately 9 mg, 18 mg and 37 mg of calcium ionsrespectively, representing 30 mg, 60 mg and 120 mg of calcium per grampectin.

It is generally understood that 30 mg of calcium ions is sufficient tofully gel 1 gram of amidated low ester pectin, so it is likely thatadding two and four times more calcium causes some contraction andpartial collapse of the originally gelled pectin structure, therebyresulting in syneresis, i.e., the release of water. In any event, thesurprising discovery that a methanolic solution of calcium lactate isable to uniformly dry and coat a Petri dish surface, where the resultingdeposited and dried film remains bound to a polystyrene or glass Petridish surface while the coating gradually dissolves into an overlaidpectin-containing culture medium could not have been predicted.Simultaneous sterilization of the container and solution was also asurprising result. The calcium lactate deposited from evaporation ofmethanol was surprisingly stable (for many weeks) upon the containerwalls. The ability to store the calcium lactate coated container forlong periods of time and subsequently add the liquid growth media to thecontainer was unanticipated.

The embodiment of a self-contained kit containing a package ofpectin-containing growth media, a package of calcium lactate (or otherorganic salt of calcium) in methanol, and a pre-sterilized Petri dishenables another aspect of durability, transportation, and storage. Ifneeded, the calcium lactate in methanol can be used to sterilize thecontainer prior to evaporation of the methanol. The deposited calciumlactate is sterilized by the methanol during application.

It was subsequently found that a significant portion of the methanolsolvent for anhydrous calcium lactate could be replaced by a significantportion of other miscible alcohols including but not limited to ethanoland isopropanol. These higher molecular weight alcohols are poorsolvents for calcium lactate but because methanol is a very effectivesolvent for calcium lactate, 50% or more of the methanol may be replacedby these higher alcohols and calcium lactate will still dissolve.However, such mixed alcohol solvents evaporate more slowly than methanolalone so that a longer time interval is generally required for thecalcium lactate film to dry. As a point of reference if 1.3 mL of 100%methanol containing 50-200 mg calcium lactate is applied to a 15 mm×90mm Petri dish surface at room temperature, as little as 10-15 minutesare required to fully dry the film from the applied solution dependingin part upon the ambient temperature and the relative humidity. Inpreferred embodiments, some water is in the calcium lactate/methanolsolution, causing the subsequent calcium lactate film to have a whitishtranslucent appearance.

The present technology is not limited to the solvents discussed herein,but methanol surprisingly provided sterilization along with evendeposition of the calcium lactate on the container walls.

The present technology is not limited to calcium lactate, and anyorganic calcium salt that dissolves or partially dissolves in a rapidlyevaporating solvent is encompassed in the system and methods/embodimentsdisclosed by the present technology. The salts useful for the presenttechnology are, in general, moderately soluble in water allowing a thincoating of the salts on a cell culture container to gradually dissolveinto a cell growth medium applied over the salt coating, whilepreferably being highly soluble, or moderately soluble in volatilesolvent(s). In some examples a slurry of calcium salt is sufficient inwhich case the organic calcium salt can be highly, moderately or poorlysoluble in the volatile solvent. While the volatile solvent used can beother than methanol, some examples of other organic calcium salts are:calcium acetate, calcium malate, calcium citrate malate, calciummaleate, calcium lactate gluconate, calcium citrate salts combined withother salts, calcium fumarate, calcium gluconate, calcium propionate,calcium stearoyl-2-lactylate, calcium galactonate, calcium ascorbate,and combinations thereof and/or hydrates/anhydrous forms. Calciumfumarate anhydrous, calcium malate anhydrous, and calcium gluconate weretested on Petri dishes, using methanol slurries for application, andthese salts smoothly gelled the pectin-containing growth medium inlonger times than calcium lactate. The present technology encompassesany calcium salt that is highly or moderately soluble in a volatilesolvent, for example, methanol, with or without additional calciumlactate. The present technology does not encompass non-organic salts ofcalcium that are highly soluble in water but not in volatile organicsolvent(s). Preferably the calcium salt, such as calcium lactate, usedto form the calcium salt film is moderately soluble in the gellablegrowth medium added to the culture container, and/or moderately solublein water (i.e., the salt is not highly soluble in water, i.e., ispreferably not calcium chloride, calcium acetate, or calcium nitrate,and preferably is not poorly soluble in water such as calcium phosphatedibasic or calcium carbonate), so as to slowly and gradually releasecalcium ions into the gellable growth medium consistent with theformation of a thin, uniform film of calcium salt on the growth surfaceof the container, which allows uniform gelation of the culture medium. Acalcium salt film that is formed from a highly water soluble orheterogeneously distributed calcium salt can result in too fast arelease of calcium ions into the gellable culture medium, producing aheterogeneous or poorly gelled culture medium. While these examples arenot intended to limit the scope of the present technology, othercombinations of volatile solvents and calcium ions are envisioned andwithin the scope of the present technology, which provides the systemand methods necessary to include the abovementioned benefits and/orembodiments.

Another embodiment of the present technology is a system ofmanufacturing the sterile pectin-containing culture media and packagingthe media in the correct amounts/concentrations for use in a Petri dishor similar container by an end user who has limited skills. This aspectof the technology is combined with a system of manufacturing a containerof calcium lactate solution in methanol that is pre-sterilized andsecurely packaged in the correct concentration/amount to gel the packageof pre-sterilized pectin-containing growth media. The system ofmanufacturing includes calculating the concentrations and amounts ofpectin culture media and calcium lactate solution such that the end userdoes not need to consider such skills. The internal surface area of thePetri dish or container to be included in the kit is included in thecalculation of the volume of calcium lactate solution packaged in thekit. The volume and concentration of calcium lactate is calculated sothat gelling of the growth medium is done without the end user needingto consider these factors. This system enables quality production of akit containing the sealed packages described above, a sterile Petri dishor similar container with instructions. The kit can be sealed andstored, shipped, or immediately used. Various kits can be provideddepending on the intended organism to be grown upon the growth media inthe kit.

In yet another embodiment of the present technology, sterilization ofthe entire kit and contents is performed after assembly of the kit by,for example, irradiation or microwaves. In this example the kit andcontents are sterilized before shipment to the end user.

Another aspect of the present technology is a method of using the kit.For example, the end user, who may be a person of ordinary skill, opensthe kit, opens the Petri dish, and opens the container of calciumlactate solution. The end user places the Petri dish on a level surfaceand pours the calcium lactate solution into the Petri dish, nextensuring the Petri dish has an evenly distributed coating of thesolution at least entirely upon the bottom interior surface. The enduser waits a brief time for the methanol to evaporate, thereby formingan even coating of calcium lactate at least upon the bottom interiorsurface of the Petri dish (see FIG. 1). The end user then opens thepackage of sterile, pectin-containing culture medium and pours themedium into the Petri dish. The lid to the Petri dish is thenimmediately applied. After waiting a brief period of time, e.g., 15-30minutes, the pectin-containing culture medium forms a gel. The end userthen follows instructions for sampling and/or placing a test specimenonto the sterile growth medium.

If, for example, the end user is provided a sterile culture containerwith an pre-applied internal coating of calcium lactate, the end usercould be instructed to add the pectin-containing growth media and awaitgelling.

The methods described above are not limited by the examples provided.For example, the calcium lactate solution can be provided in a breakablepackage within a container with a flexible pressure point, and the enduser merely applies pressure to the breakable package, releasing thecalcium lactate solution. After methanol evaporation, the growth mediacan be applied to the calcium lactate film.

Instead of calcium lactate, a slurry/suspension of a different organicsalt of calcium can be used, for example, a slurry of a fine powder ofcalcium fumarate anhydrous, calcium malate anhydrous, or calciumgluconate. When a slurry is used, the end user tilts a Petri dish (orother container) containing the slurry back and forth to uniformly coatthe bottom of the dish and then allows the slurry to dry. After drying,approximately 15 mL of a culture medium is poured into a 90 mm diameterPetri dish and a pectin-containing cell growth medium may gel over alonger period of time compared to the gel-time for calcium lactate. Thecalcium salt coatings formed in Petri dishes or other containers frommethanolic suspensions/slurries are not as uniform as the coatingsformed from calcium lactate but are adequate for the purpose of evenlygelling a culture medium (e.g., see FIGS. 2 and 3 compared to FIG. 1).

Petri Dish or Container Selection

The Petri dish or container can be disposable or reusable and someexamples of suitable materials include polystyrene resin, polylactideresin, or glass. The Petri dish or container can be made of variousrigid materials such that a shallow cylindrical or other shaped dish isprovided with an optional lid. The Petri dish or container should berelatively inert with respect to the alcohol and solutes added so thatstructural integrity is maintained. The shape of the container can bechanged to suit the growth conditions or sampling conditions. Thecontainer is typically transparent, but opaque or translucent materialsare encompassed because, for example, a light sensitive organism couldbe growing on the culture medium. The lid could be configured forspecific environmental sampling or configured to enable organism growthupward. The present technology is not limited by the composition of thePetri dish or container, and the examples provided herein are forillustrative purposes only. Pre-sterilized Petri dishes can be purchasedfor use in manufacturing/providing the kits described herein.

In some examples, the Petri dish or container may be made of glass orborosilicate, ethylene vinyl acetate (EVA) polymers, ethylene methylacrylate (EMA) polymers, polyethylenes [including, for example, lowdensity polyethylene (LDPE), linear low density polyethylene (LLDPE),high density polyethylene (HDPE), and ultra-high molecular weightpolyethylene (UHMWPE), polypropylenes, ethylene-propylene rubbers,ethylene-propylene-diene rubbers, polystyrene, poly (1-butene),poly(2-butene), poly(l-pentene), poly(2-pen-tene),poly(3-methyl-1-pentene), poly(4-methyl-1-pentene),1,2-poly-1,3-butadiene, 1,4-poly-1,3-butadiene, polyiso-prene,polychloroprene, poly(vinyl acetate), poly(vinylidene chloride),poly(vinylidene fluoride), poly(tetrafluoroethyl-ene), and the like, andcombinations thereof. The lid of the Petri dish or container canoptionally be made of similar material or designed for specificsampling/growth purposes.

In other examples, the Petri dish or container may be made of polymersincluding olefin homopolymers and copolymers (especially, polyethylenes,polypropylenes, ethylene vinyl acetate polymers, and combinationsthereof). Other polymers include olefin homopolymers and combinationsthereof (polyethylenes and combinations thereof; ultra-high molecularweight polyethylenes (UHMWPE) and combinations thereof). The lid of thecontainer or Petri dish can be made of a material different from the lidor of various materials.

In alternative examples, the Petri dish or container is not rigid and isflexible so that the calcium lactate solution and/or growth media can becontained inside of the flexible container. In these examples, thecalcium lactate solution and/or growth media is released from abreakable package within the flexible container package.

In some embodiments, the Petri dish or container is specially designedin shape/configuration to trap organisms and/or to prevent entry ofcertain organisms.

FIG. 1 shows a photo of a 90 mm diameter polystyrene Petri dish with aninterior film coating of calcium lactate formed and deposited byevaporation of a solution containing 1 mL methanol, 100 mg calciumlactate anhydrous and 4% by weight water, in which the resulting coatingis suitable for gelling a pectin-containing growth medium. The Petridish was tilted back and forth to uniformly coat the bottom of the dishand then allowed to dry on a flat surface.

FIG. 2 shows a photo of a 90 mm diameter polystyrene Petri dish with aninterior coating of calcium malate powder deposited by evaporation of aslurry containing 1 mL methanol and 100 mg calcium malate anhydrousparticles, in which the resulting coating is suitable for gelling apectin-containing growth medium. The Petri dish was tilted back andforth to uniformly coat the bottom of the dish and then allowed to dryon a flat surface.

FIG. 3 shows a photo of a 90 mm diameter polystyrene Petri dish with aninterior coating of calcium gluconate powder deposited by evaporation ofa slurry containing 1 mL methanol and 200 mg calcium gluconate anhydrousparticles, in which the resulting coating is suitable for gelling apectin-containing growth medium. The Petri dish was tilted back andforth to uniformly coat the bottom of the dish and then allowed to dryon a flat surface.

Definitions

As used herein “methanolic” solutions are any solutions containing atleast some methanol ranging up to solutions containing mostly orentirely methanol, such as solutions that contain 5-100 volume % ofmethanol and the remainder of water or another miscible solvent that canbe removed by evaporation or other convenient mechanism. Likewise, themethanol slurries disclosed herein are slurries that may contain atleast some methanol ranging up to slurries containing mostly or entirelymethanol. A “solution comprising calcium lactate and methanol” may alsocontain any amount of water or other ingredients. Slurries of organicsalts of calcium in methanol may also contain any amount of water orother ingredients.

As used herein “growth media” and “growth medium” can be any mixturethat sustains or nourishes growth of one or more organisms such asbacteria, molds or viruses and the mixture may be designed to preventgrowth of other organisms.

As used herein “excessive syneresis” (release of free liquid from thegel) refers to release of free liquid to such an extent that it mayinterfere with some experiments involving microorganisms, generallyundesirable for measurements, yet it could potentially be considerednon-interfering for other biological growth conditions, for example, forsome eukaryotic cells. While excessive syneresis is not desirable fortraditional bacterial/fungal colony growth in Petri dishes, excessivesyneresis might be desirable for growth of some other biologicalorganisms, so the definition is not construed so as to limit the presenttechnology in the scope of different gels formed or differentingredients used.

As used herein “Petri dish” or “container” describes a container thatwill contain a coating of a suitable calcium salt such as calciumlactate after evaporation of a calcium lactate solution in methanol orsimilar volatile solvent. The Petri dish or container can be flexible orrigid, opaque, transparent, or translucent. The Petri dish or containermay have a lid or be entirely self-contained, without lid. The surfacearea of the container should be calculable at one static conditionbecause the amounts and concentrations of the calcium lactatesolution(s) described herein must be calculated for appropriate usagewith the container and simultaneously calculated for appropriate gellingof the culture growth media, but the container can be expandable withchanging surface area(s). Thus, the container can be inflatable orcompressible and can change shape if needed.

As used herein “volatile solvent” includes any organic solvent that,when 1 mL is placed into the bottom of an open dry Petri dish withapproximately 90 mm diameter, will evaporate to dryness in less than 1hour or even a substantially shorter time such as 10-20 minutes at atemperature of 25° C. and a relative humidity of 10%.

As used herein “highly soluble” refers to a solute that is soluble in asolvent at greater than or equal to 10% by weight. “Moderately soluble”refers to a solute that is soluble in a solvent at from 1% to less than10% by weight. “Poorly soluble” refers to a solute that is soluble in asolvent at less than 1% by weight. All solubilities are as determined at25° C.

As used herein a “slurry” is a suspension of a fine powder in a solvent,wherein the powder may be either highly, moderately or poorly soluble inthe solvent.

As used herein any other organic salt of calcium can be substituted inplace of the term “calcium lactate” as other organic salts of calciumare within the scope of the present technology and several have beentested with success (FIGS. 2 and 3).

Those with skill in the art would readily appreciate that the presenttechnology is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The systems, methods, usages, kits,variances, and compositions described herein as presently representativeof preferred embodiments are exemplary and are not intended aslimitations on the scope of the technology. Changes therein and otheruses will occur to those skilled in the art, which are encompassedwithin the ideas of the technology, are defined by the scope of theclaims. Changes envisioned but not fully discussed are providing thepresent technology with a means of heating or cooling for example hotpacks or cold packs so that the present technology may be used in coldor hot environments, providing the present technology with an attractantthat attracts specific organisms, or alternatively a repellant. Someother examples not fully discussed are modifications to the kit enablingusage in extreme environments.

The technology illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the technology claimed. Thus, it should be understood thatalthough the present technology has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this technology as defined by the appended claims.

In addition, where features or aspects of the technology are describedin terms of kits, groups or other grouping of alternatives, for examplepackages, methods, containers, lids, order of sterilization, thoseskilled in the art will recognize that the technology is also therebydescribed in terms of any individual member or subgroup of members ofthe methods, kit, group, containers, lids, orders of sterilization orother alternative grouping of the kits, methods, and physicalembodiments.

Also, unless indicated to the contrary, where various numerical valuesor value range endpoints are provided for embodiments, additionalembodiments are described by taking any 2 different values as theendpoints of a range or by taking two different range endpoints fromspecified ranges as the endpoints of an additional range. Such rangesare also within the scope of the described technology. Further,specification of a numerical range including values greater than oneincludes specific description of each integer value within that range.

Thus, additional embodiments are within the scope of the technology andwithin the following claims while the concepts of the systems, methods,and kits described herein are broadly applicable to moving the provisionof biological culture media into farther reaches.

What is claimed is:
 1. A method for preparing a gelled biological growth medium, the method comprising: a) providing a container for culturing microorganisms and a solution comprising calcium lactate and methanol; b) depositing the solution onto a growth surface of the container; c) allowing the solution to evaporate, thereby forming a calcium lactate precipitate on the growth surface; d) coating the calcium lactate precipitate with a liquid growth medium comprising low methoxyl pectin; and e) allowing the liquid growth medium to gel, thereby forming the gelled biological growth medium.
 2. The method of claim 1, wherein the low methoxyl pectin is a low methoxyl amidated pectin and is the sole gelling agent.
 3. The method of claim 1, wherein the low methoxyl pectin has a methoxyl content of from about 2% to about 8% by weight.
 4. The method of claim 3, wherein the low methoxyl pectin has a methoxyl content from about 5% to about 6%.
 5. The method of claim 1, wherein the liquid growth medium comprises from about 10 to about 30 grams of pectin per liter.
 6. The method of claim 1, wherein the container is a thermoplastic or glass Petri dish.
 7. The method of claim 6, wherein the Petri dish comprises polystyrene or polylactide.
 8. The method of claim 1, wherein the solution comprising calcium lactate and methanol comprises calcium from about 10 to about 60 milligrams of calcium per gram of low methoxyl pectin present in the liquid growth medium.
 9. The method of claim 1, wherein the calcium lactate precipitate comprises calcium from about 10 to about 60 milligrams of calcium per gram of low methoxyl pectin present in the liquid growth medium.
 10. The method of claim 1, wherein the solution comprising calcium lactate and methanol further comprises between about 2% and 5% water by volume.
 11. The method of claim 1, wherein the liquid growth medium is selected from the group consisting of potato dextrose broth, yeast extract dextrose broth, and combinations thereof.
 12. The method of claim 1, wherein the liquid growth medium further comprises an antimicrobial agent that selectively inhibits growth of one group of microbes while permitting growth of another group of microbes.
 13. The method of claim 12, wherein the liquid growth medium comprises from about 50 to 100 micrograms per milliliter of chloramphenicol to inhibit growth of bacteria while permitting growth of yeast and mold.
 14. The method of claim 1, wherein the container is sterilized by the methanol.
 15. The method of claim 1, wherein the container is a Petri dish, and wherein the solution comprising calcium lactate and methanol is added to in an amount from about 0.5 mL to 2 mL per 64 cm² area of the growth surface.
 16. The method of claim 1, wherein the solution comprising calcium lactate and methanol comprises from about 25 to about 100 volume % methanol.
 17. The method of claim 1, wherein the solution comprising calcium lactate and methanol comprises from about 85 mg to about 350 mg of anhydrous calcium lactate per gram of the low methoxyl pectin. 